Mri contrast agents endowed with concentration independent responsiveness

ABSTRACT

The present invention relates to a method for the in vivo, ex vivo, or in vitro determination of physical or chemical parameters of diagnostic interest by use of a slow tumbling paramagnetic agent that is responsive to changes of said physical or chemical parameter through changes in the R 2p /R 1p  ratio allowing the determination of the said parameter in a manner that is independent on the actual agent concentration.

The present invention relates to the field of diagnostic imaging by useof Magnetic Resonance Imaging techniques (MRI). More in particular, itrelates to a new use of a class of paramagnetic contrast agents in amethod for the in vivo determination of physical or chemical parametersof diagnostic interest, independently from the local concentration ofthe administered contrast agent.

BACKGROUND OF THE INVENTION

It is now well established that the potential of Magnetic ResonanceImaging (MRI) procedures can be further enhanced when this diagnosticmodality is applied in conjunction with the administration of contrastagents (CAs), i.e. chemicals able to promote marked changes in therelaxation rates of the tissue protons. The MRI CAs are principallyrepresented by paramagnetic complexes, mostly containing Gd(III),Fe(III) or Mn(II) ions, which affect the relaxation rates of the bulkwater through the exchange of the water molecules in their coordinationspheres (Caravan P, et al. Chem Rev 1999, 99:2293-2352; the Chemistry ofContrast Agents in Medical Magnetic Resonance Imaging. Chichester, UK:John Wiley & Sons; 2001. p 45-120).

The efficacy of a paramagnetic complex is assessed by its protonrelaxation enhancement or relaxivity (r_(i), i=1,2), which representsthe increase of the proton relaxation rate of an aqueous solutioncontaining 1 mM concentration of the paramagnetic agent in comparison tothe proton relaxation rate of neat water. For a paramagnetic complex,the proton relaxation enhancement is chiefly governed by the choice ofthe paramagnetic metal, the rotational correlation time of the complexand the accessibility of the metal to the surrounding water molecules,i.e. the rapid exchange of water with the bulk.

Two characteristic relaxation rates are involved: R₁ that is defined asthe inverse of the longitudinal relaxation time or spin latticerelaxation time T₁, i.e. 1/T₁, and R₂ that is defined as the inverse ofthe transverse relaxation time or spin-spin relaxation time T₂, i.e.1/T₂.

The higher the longitudinal relaxivity (r₁), the larger is the signalenhancement detected in the corresponding T₁-weighted MR images and thebetter is the contrast differentiation in the resulting images.

Some contrast agents exist which relaxivity is related to and may dependupon the physical or chemical characteristics of the microenvironment inwhich they distribute. These agents are known as responsive agentsbecause the contrast, in the image they promote, is responsive to aphysical or chemical parameter of diagnostic interest. Several systemshave been reported which relaxivity is made dependent on pH,temperature, PO₂, enzymatic activity, ion and metabolite concentrations(Jacques V, Top Curr Chem 2002, 221, 123-164).

However, such a peculiar responsive property could not have beenproperly exploited in practice because the detected T₁-variations cannotbe unambiguously ascribed to a change in relaxivity and, consequently,to a variation on the physical or chemical parameter under examination,if the actual concentration of the paramagnetic complex is unknown. Tobe effective, a MRI responsive agent should display its responsivenessin a concentration independent mode. Accordingly, it is an object of thepresent invention a MRI method and agents allowing the overcoming ofthis drawback.

SUMMARY OF THE INVENTION

In accordance with the above object, the present invention is directedto the identification of a class of responsive agents which use allowsthe measurement of a physical or chemical parameter of diagnosticinterest independently on the actual concentration of the agent itself.In a different aspect, the invention relates to a method of use of saidagents for the determination of a physical or chemical parameter ofdiagnostic interest, independently on the absolute concentration of theadministered agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 relates to calculated R_(2p)/R_(1p) values as a function of themagnetic field strength for a macromolecular Gd(III) complex (q=1, r=3Å, τ_(M)=200 ns) endowed with τ_(R) values in the 0.5-10 ns range. Forthe electronic relaxation, a Δ² value of 2·10¹⁹ s⁻² and a τ_(V) value of10 ps (corresponding to τ_(1S) values in the 0.65-500 ns interval) havebeen used.

FIG. 2 a) relates to calculated R_(2p)/R_(1p) values as a function ofτ_(R) for Gd(III)-based systems endowed with τ_(M) values in the 0.05-1μs range at 7 T.

FIG. 2 b) relates to calculated R_(2p)/R_(1p) values as a function ofτ_(M) for Gd(III)-based systems endowed with τ_(R) values in the 0.5-10ns range at 7 T.

FIG. 3 shows the structure of the (Gd-1)₄/Avidin adduct.

FIG. 4 shows the temperature dependence of the R_(2p)/R_(1p) ratio forthe (Gd-1)₄/Avidin adduct at 7 T. The formula of the Gd-1 complex isincluded in the experimental section. The reported bars refer to thestandard deviation of three measurements of solutions containing 0.125,0.25, and 0.5 mM of the paramagnetic complex.

FIG. 5 reports the pH dependence of the R_(2p)/R_(1p) ratio for theGd-DOTP/polyarginine adduct (molar ratio=10) at 14.1 T and 312 K. Theconcentration of the Gd(III) complex was of 0.5 mM (filled squares) and0.25 mM (open squares).

FIG. 6 shows the structure of the macromolecular adduct Gd-II.

FIG. 7 reports the magnetic field dependence of R_(1p) (filled symbols)and R_(2p) (open symbols) measured at 25° C. and 600 MHz for a solutioncontaining the Gd-II adduct ([Gd]=1 mM). pH 7 (squares), pH 10(circles), and pH 12 (triangles).

FIG. 8 reports the dependence of the R_(2p)/R_(1p) ratio on theconcentration of Gd(III) for the Gd-II macromolecular adduct at four pHvalues: pH 7 (squares), pH 8.5 (circles), pH 10 (triangles), and pH 12(diamonds) (600 MHz. 25° C.).

FIG. 9 shows the corresponding pH dependence of the R_(2p)/R_(1p) ratiocalculated from the data point reported in FIG. 8.

FIG. 10 reports the temperature dependence of R_(1p) (circles) andR_(2p) (squares) measured at 300 MHz for a solution containingparamagnetic liposomes (POPC/Chol/DSPE-PEG, molar ratio 55:40:5)entrapping GdHPDO3A 200 mM. The total concentration of the Gd(III)complex in the suspension was 6.6 mM.

FIG. 11 shows the dependence of the R_(2p)/R_(1p) ratio on theconcentration of liposomes (bottom x-axis) and Gd(III) (top x-axis) forthe paramagnetic liposomes described in Example 4. Temperatures: 298 K(squares), 302 K (circles), 307 K (upper triangles), and 312 K(diamonds) (300 MHz).

DESCRIPTION OF THE INVENTION

A class of contrast agents that solve the problem faced by the presentinvention is represented by responsive paramagnetic systems that areresponsive to a physical or chemical parameter of diagnostic interest interms of affecting T₁ and T₂ of the solvent water protons, and in whichsuch T₁ and T₂ responsiveness to said parameter follows differentbehavioral functions. For these responsive systems the R_(2p)/R_(1p)ratio may be made independent upon the actual concentration of the agentitself through maintaining the functional dependence thereof to theparameter of interest. Accordingly, by use of these systems, adetermination of the parameter of interest may be obtained asindependent on the actual agent concentration through the measurement ofsuch R_(2p)/R_(1p) ratio.

In the present invention, unless otherwise indicated, with physical orchemical parameter of diagnostic interest we intend a parameter selectedfrom: temperature, pH, partial pressure of oxygen (PO₂) or carbondioxide (PCO₂), specific ion or metabolite concentration, or specificenzymatic activity.

By knowing the value of the said physical or chemical parameter(s), askilled man in the art may easily provide diagnostic evaluations of anyphysiological or metabolic process of diagnostic interest relying on thesaid parameter(s).

For protons dipolarly coupled to a paramagnetic center, at magneticfield strengths higher than 0.2 T, the R_(2p) and R_(1p) terms (whereinR_(2p) is the paramagnetic contribution to the measured transverserelaxation rate and R_(1p) is the paramagnetic contribution to themeasured longitudinal relaxation rate) in the presence of amacromolecular Gd(III) complex are described by the following equations:

$\begin{matrix}{R_{1_{P}} = {{\frac{P_{M}}{T_{1\; M} + \tau_{M}}\frac{1}{T_{1\; M}}} = {\frac{6}{15}\frac{K^{DIP}}{r_{H}^{6}}( \frac{\tau_{C}}{1 + {\omega_{H}^{2}\tau_{C}^{2}}} )}}} & (1) \\{R_{2_{P}} = {{\frac{P_{M}}{T_{2\; M} + \tau_{M}}\frac{1}{T_{2\; M}}} = {\frac{1}{15}\frac{K^{DIP}}{r_{H}^{6}}( {{4\; \tau_{C}} + \frac{3\; \tau_{C}}{1 + {\omega_{H}^{2}\tau_{C}^{2}}}} )}}} & (2)\end{matrix}$

where P_(M) is the molar fraction of the mobile protons dipolarlyinteracting with Gd(III) ion (e.g. for one water molecule in the innercoordination sphere of GD(III), P_(M) is equal to

$\frac{q\lbrack{GdL}\rbrack}{55.6}$

with q=number of water molecule coordinated to Gd(III)), τ_(M) is theirmean residence lifetime, r_(H) their mean distance from the metalcenter, ω_(H) their Larmor frequency (rad·s⁻¹), and τ_(C) theirmolecular correlation time (τ_(C) ⁻¹=τ_(M) ⁻¹+τ_(R) ⁻¹+τ_(1S) ⁻¹ withτ_(R)=rotational correlation time or reorientational time andτ_(1S)=longitudinal electronic relaxation time). K^(DIP) is a constantvalue (3.887·10⁻⁴² m⁶·s⁻²) related to the dipolar interaction betweenthe electron and the nuclear spins.

From equations 1-2 it may be derived that for said macromolecular agentthe R_(2p)/R_(1p) ratio is independent on the concentration of the agentitself, being only affected by τ_(M), τ_(R), τ_(1S) and ω_(H) values.Accordingly, for these compounds, changes in the R_(2p)/R_(1p) ratioacting as a reporter of a change in the parameter of interest are onlyrelated to a change in τ_(R) and/or τ_(M) and/or τ_(1S) where suchchanges have a different effect on R_(2p) and R_(1p) values.

Therefore, a first object of the present invention is the use of a slowtumbling paramagnetic agent responsive to changes of a given physical orchemical parameter of diagnostic interest through changes inR_(2p)/R_(1p) ratio for the preparation of diagnostic compositions forthe determination of the said parameter in a human or animal body organ,fluid or tissue, by use of the MRI, in a manner which is independent onthe absolute concentration of said administered agent.

A further object of the present invention is a method for the in vivo,or in vitro or ex vivo determination, by use of MRI, of a physical orchemical parameter of diagnostic interest in a human or animal bodyorgan, fluid or tissue, which method comprising:

-   -   administering to said human or animal a diagnostically effective        amount of a slow tumbling paramagnetic agent that is responsive        to microenvironmental changes of said parameter through changes        in the R_(2p)/R_(1p) ratio, and    -   recording a MRI imaging responsive for said parameter, that is        independent on the actual concentration of the administered        agent, through the measurement of said ratio.

The term “diagnostically effective amount” as used herein, refers to anyamount of the slow tumbling paramagnetic agent of the invention, orpharmaceutical composition thereof, that is sufficient to fulfil itsintended diagnostic purpose(s), i.e. to provide highly contrasted anddiagnostically effective images enabling the determination of theparameter of interest.

The said dosages are obviously selected by the health professionaldepending on the administered slowly mowing agent.

According to the present invention with “slow tumbling paramagneticagent or system” as used herein interchangeably, we intend aparamagnetic agent or system having a τ_(R) value ≦1 ns.

In one preferred aspect of the invention the said slow tumblingparamagnetic agent is a macromolecular paramagnetic metal complex.

In another aspect, the said slow tumbling paramagnetic system isrepresented by a paramagnetic system in which the relaxation enhancementis generated by magnetic susceptibility effects.

Such effects, which are strictly limited to the transverse evolution ofthe nuclear magnetization (i.e. they only affect T₂ values), are inducedby microscopic magnetic field gradients created by magneticsusceptibility differences between compartments (J L Boxerman, R MWeisskoff, B R Rosen “Susceptibility effects in Whole Body Experiments”in “Methods in Biomedical Magnetic Resonance Imaging and Spectroscopy”Ed. I R Young, John Wiley & Sons, Chichester, Vol. 1, pp 654, 2000).

This T₂-specific contribution is directly dependent on the square of theintensity of the magnetic field gradients that are proportional to themagnetic susceptibility differences and the magnetic field strength, andthey are affected by the relative size of the compartments. Thedifference of the magnetic susceptibility between the compartments canbe significantly enhanced if a rather high concentration of aparamagnetic species is confined in one compartment. Typical examples ofslow tumbling paramagnetic systems generating a significantsusceptibility T₂-effect are represented by nano-sized compartmentscontaining rather high amount of a paramagnetic metal complex.

According to the present invention, unless otherwise indicated, with theterm “nano-sized systems” we intend a system having a mean diameter ≧5nm. Preferably, the said systems entrap the paramagnetic metal complexat a minimum concentration of 5 mM.

A non-exhaustive list of nano-sized systems that can entrap paramagneticmetal complexes includes, for instance, nanoparticles, microemulsions,liposomes, protein cavities, and the like.

In other words, as for all the macromolecular paramagnetic metalcomplexes according to the invention, for the said nano-sized systemsthe R_(2p)/R_(1p) ratio is independent on the concentration, being itonly affected by the magnetic field strength, the amount of theentrapped complex and the size of the compartment. In an embodiment ofthe invention, accordingly, the slow tumbling paramagnetic agentdisplaying a R_(2p)/R_(1p) ratio responsive to microenvironmentalchanges of a physical or chemical parameter of diagnostic interest isrepresented by a nano-sized system which transverse relaxation rate isaffected by magnetic susceptibility effects for which, moreover, theR_(2p) value, and consequently the R_(2p)/R_(1p) ratio, is dependent ona physical or chemical parameter or a physiological or metabolic processof diagnostic interest.

Besides the selective T₂-effect arising from magnetic susceptibilityeffects, the R_(2p)/R_(1p) ratio for a nano-sized system could be madedependent on the parameter of interest by acting on R_(1p). Forinstance, this goal may be achieved if the physico-chemical parameteraffects the water permeability of the compartment that entraps theparamagnetic complex.

Thus, in one embodiment, the present invention relates to a method forthe in vivo, or in vitro or ex vivo determination, by use of MRI, of aphysical or chemical parameter of diagnostic interest in a human oranimal body organ, fluid or tissue in which a nano-sized systementrapping a paramagnetic metal complex is administered that isresponsive to changes of a given physical or chemical parameter or aphysiologic or metabolic process of diagnostic interest through changesin R_(2p)/R_(1p) ratio.

As above said, for the particular class of slow tumbling paramagneticagent of the invention, the R_(2p)/R_(1p) ratio is independent on theactual concentration of the agent itself, while the said ratio is onlyaffected by τ_(M), τ_(R), τ_(1S), size, water permeability of thecompartment, and ω_(H) values.

So, from a different point of view, it may be said that the measuredR_(2p)/R_(1p) ratio for the said agents acts as a reporter of a changein the parameter of interest because the changes that the said parameterpromote in τ_(R) and/or τ_(M) and/or τ_(1S) values, and/or size, and/orwater permeability of the compartment, at a given ω_(H) value, have adifferent effect on R_(2p) and R_(1p) values.

In particular, when the slow tumbling paramagnetic system used in themethod of the invention is a macromolecular paramagnetic metal complex,the measured R_(2p)/R_(1p) ratio acts as a reporter of a change in theparameter of interest because the changes that the said parameterpromotes in τ_(R) and/or τ_(M) and/or τ_(1S) values, at a given ω_(H)value, have a different effect on R_(2p) and R_(1p) values.

When, in a different embodiment, the administered slow tumblingparamagnetic system is a nano-sized system entrapping a paramagneticmetal complex, the measured R_(2p)/R_(1p) ratio acts as a reporter of achange in the parameter of interest because the changes that the saidparameter promotes in the size, and/or water permeability of thecompartment have a different effect on R_(2p) and R_(1p) values. Theratiometric method for responsive agents as per the present inventionrelies, accordingly, on the exploitation of the differential effect thatthe correlation times τ_(R), τ_(M) and τ_(1S) have on R_(1p) and R_(2p)values for slowly tumbling paramagnetic metal complexes.

In a different aspect, the said ratiometric method may further relies onthe exploitation of the specific effect on R_(2p) values caused by thepresence of magnetic susceptibility effects when the slow tumblingparamagnetic systems is a nano-sized systems entrapping a paramagneticcomplex.

With the aim to define how to suitably select such τ_(M), τ_(R), τ_(1S)and ω_(H) values and, consequently, how to define the macromolecularparamagnetic metal complex that may advantageously be used in the methodof the invention, a first theoretical simulation based on Eqs. 1-2 hasbeen performed showing that, at the magnetic field strengths used in MRI(0.2-7 T), the R_(2p)/R_(1p) ratio begins to be sensitive to therotational mobility of the Gd(III) complex (τ_(R)) for τ_(R) valueslonger than 0.5 ns, wherein τ_(R) values longer than 1 ns are preferredand values longer than 5 ns are even more preferred (FIG. 1).

Once the magnetic field strength is settled, τ_(R) and τ_(M) values havean opposite effect on the R_(2p)/R_(1p) ratio as it may be derived bylooking at the additional simulations performed and reported in FIG. 2a) and b). In the simulation of FIG. 2 a) the R_(2p)/R_(1p) values havebeen reported as a function of τ_(R) for Gd(III)-based systems endowedwith τ_(M) values in the 0.05-1 μs range at 7 T. In the simulation ofFIG. 2 b) R_(2p)/R_(1p) values have been calculated as a function ofτ_(M) for Gd(III)-based systems endowed with τ_(R) values in the 0.5-10ns range at 7 T. From equations 1 and 2 it follows that τ_(R) affectsthe R_(2p)/R_(1p) ratio through its effect on τ_(C) and consequently onT_(iM) (i=1,2), whereas τ_(M) influences R_(2p)/R_(1p) through thelimiting effect on Rip via the (T_(iM)+τ_(M)) term.

Based on these results, a preferred class of responsive agents for usein the method of the invention may comprise macromolecular paramagneticcomplex compounds endowed with both dipolarly coupled protons and τ_(R)value ≧1 ns, provided that their rotational mobility (τ_(R)) and/or themean residence lifetime τ_(M) of the mobile protons dipolarly coupled totheir metal center and/or their longitudinal electronic relaxation timeτ_(1S) are dependent on the parameter of interest. For a given compound,it is well known in the art that the dependence of the said τ_(R),τ_(M), τ_(1S) values on the parameter of interest may be easily verifiedby plotting of the R_(2p)/R_(1p) ratio over the said parameter: when asignificant variation of the said ratio over the parameter of interestis verified, then τ_(R) and/or τ_(M) and/or τ_(1S) of the testedcompound are dependent on this parameter.

Among the said responsive agents, particularly preferred aremacromolecular paramagnetic metal complex compounds endowed with τ_(R)values from 1 to 10 ns and τ_(M) values from 0.01 to 1 μs.

In the present description, unless otherwise indicated, with the termmacromolecular paramagnetic complex it has to be intended a paramagneticmetal complex endowed with slow tumbling rate, that is to say a complexendowed with a τ_(R) value longer than 0.5 ns, wherein τ_(R) valueslonger than 1 ns are preferred and values from 5 to 10 ns are even morepreferred.

The necessary slow tumbling rate of the responsive agents according tothe invention may be obtained by controlling the molecular size of theparamagnetic system, for example trough the formation of covalent ornon-covalent linkages with macromolecules or endogenous substrates thatconfer to the agent the desired molecular weight.

For nano-sized responsive agents of the invention, for example, thepreferred systems are nanoparticles with a mean diameter ≧5 nm,entrapping a paramagnetic metal complex at a minimum concentration of 5mM.

The paramagnetic complex entrapped in the said nano-sized system may beany paramagnetic metal complex of the art, without any limitationconcerning its molecular weight.

As far as temperature responsive nano-sized agents is concerned, apreferred system (for imaging experiments performed at magnetic fields≧3 T) may be represented by a paramagnetic liposome endowed with thefollowing characteristics: i) mean diameter ≧150 nm, ii) waterpermeability of the liposome membrane ≧5·10⁻⁵ cm s⁻¹, iii) concentrationof the entrapped paramagnetic metal complex (referred to the inneraqueous compartment of the liposome) ≧50 mM. Preferably, the entrappedparamagnetic metal complex is a Gd(III) complex.

In the present description, with the term paramagnetic contrast agent orparamagnetic complex or paramagnetic metal complex, as used hereininterchangeably, we intend any chelated complex with a bi- and trivalentparamagnetic metal ion, preferably having atomic number ranging between20 and 31, 39, 42, 43, 44, 49, and between 57 and 83 such as, forinstance, Fe(²⁺), Fe(³⁺), Cu(²⁺), Cr(3⁺), Eu(³⁺), Dy(³⁺), La(³⁺), Yb(³⁺)or Mn(²⁺) and Gd(³⁺), this latter being even more preferred.

More preferably, the slow tumbling responsive agent for use in themethod of the invention is a macromolecular Gd(III) complex endowed witha τ_(R) value between 1 and 10 ns, a τ_(M) from 0.01 to 1 μs for which,moreover, τ_(R) and/or τ_(M) and/or τ_(1S) are dependent on theparameter of interest.

Accordingly, in a preferred method according to the invention, a slowlymoving Gd(III) complex is used having a τ_(R) value from 1 to 10 ns anda τ_(M) from 0.01 to 1 μs and wherein τ_(R) and/or τ_(M) and/or τ_(1S)are dependent on the parameter of interest.

In a equally preferred method of the invention, a slowly movingnano-sized agent is used having a mean diameter ≧5 nm and entrapping aparamagnetic complex with a concentration equal or greater than 5 mM forwhich the size of the system and/or the magnetic susceptibility effectsare dependent on a parameter of diagnostic interest.

For general, non-exhaustive, reference to the paramagnetic metalcomplexes or to the nano-sized systems entrapping a paramagnetic metalcomplex as per the invention, see, for instance, the experimentalsection below.

The paramagnetic complexes of the invention can also conveniently be inthe form of physiologically acceptable salts. Preferred cations ofinorganic bases that can be suitably used to salify the complexes of theinvention comprise ions of alkali or alkaline-earth metals such aspotassium, sodium, calcium or magnesium.

Preferred cations of organic bases comprise, inter alia, those ofprimary, secondary and tertiary amines such as ethanolamine,diethanolamine, morpholine, glucamine, N-methylglucamine,N,N-dimethylglucamine.

Preferred anions of inorganic acids that can be suitably used to salifythe complexes of the invention comprise the ions of halo acids such aschlorides, bromides, iodides or other ions such as sulfate.

Preferred anions of organic acids comprise those of the acids routinelyused in pharmaceutical techniques for the salification of basicsubstances such as, for instance, acetate, succinate, citrate, fumarate,maleate or oxalate.

Preferred cations and anions of amino acids comprise, for example, thoseof taurine, glycine, lysine, arginine, ornithine or of aspartic andglutamic acids. A further object of the present invention is adiagnostic composition for use in the MR Imaging of a physical orchemical parameter of diagnostic interest comprising at least one slowlymoving paramagnetic complex or system endowed with a τ_(R) value from 1to 10 ns and τ_(M) value from 0.01 to 1 μs for which, moreover, therotational mobility τ_(R) and/or the mean residence lifetime τ_(M) ofthe mobile protons dipolarly coupled to the metal center and/or thelongitudinal electronic relaxation time τ_(1S) are dependent on the saidparameter of interest.

In one embodiment of the invention, the said diagnostic compositioncomprises a nano-sized system entrapping a 5 mM solution of aparamagnetic complex, that is responsive to microenvironmental changesof a given physical or chemical parameter or a physiologic or metabolicprocess of diagnostic interest through changes in the R_(2p)/R_(1p)ratio and for which the size of the system and/or the magneticsusceptibility effects are dependent on a parameter of diagnosticinterest.

EXPERIMENTAL SECTION

A non-limiting list of preferred slow tumbling systems of the inventionis reported in the following section, to better exemplify the wideapplicative potential of the present invention.

Example 1

As a proof of principle, the responsive properties of a macromolecularGd(III) complex towards temperature, a parameter whose non-invasivemeasurement in vivo is important either for diagnostic or fortherapeutic purposes, have been investigated.

Among a number of possibilities, the adduct formed by four units ofcomplex 1 and Avidin was chosen as slowly moving system. Complex 1 is aderivative of GdDTPA bearing on its surface a biotin moiety. Thestructure of this complex is reported below. GdDTPA is the mostrepresentative example of MRI contrast agents, already marketed with thename Magnevist®. Avidin is a tetrameric glycoprotein (MW of ca. 68 kDa)which strongly binds (K_(A)=10¹⁵) (Green N M, Adv Prot Chem 1975, 29, 85133) four biotin units (FIG. 3).

The R_(2p)/R_(1p) protocol for the temperature measurement was tested onthree solutions containing different amounts of (Gd-1)₄/Avidin adduct(in PBS buffer at pH 7.4). The concentration of Gd-1 in the threesolutions was 0.125, 0.25, and 0.5 mM, respectively. R₁ and R₂ valuesfor water protons were measured at 7.05 T (corresponding to a protonLarmor frequency of 300 MHz) for each solution in the temperature range298-318 K.

The paramagnetic R_(1p) and R_(2p) contributions to the observedrelaxation rates were obtained by subtracting the correspondingdiamagnetic contributions (R_(id)) measured for each temperature in theabsence of the paramagnetic agent, i.e. R_(ip)=R_(i)-R_(id)).

In the investigated temperature range, the R_(2p)/R_(1p) ratio is linearwith a positive slope of ca. 0.35 units/degree (FIG. 4).

Importantly, the deviation between the R_(2p)/R_(1p) values for thethree solutions at a given temperature is very small, thus demonstratingthe validity of this approach. The increase of R_(2p)/R_(1p) values uponincreasing temperature is the result of a significant involvement ofτ_(R) in determining the transverse relaxation rate. In fact, the R_(2p)values increases with temperature, whereas R_(1p) values display anopposite tendency. This means that τ_(M) values in the range oftemperature considered are likely in the middle between T_(2M) andT_(1M) (T_(1M)>τ_(M)>T_(2M)).

Conversely, in the absence of Avidin, the R_(2p)/R_(1p) ratio forsolutions containing 0.125, 0.25, and 0.5 mM Gd-1 concentration is ca.1.3 and, moreover, it is completely unaffected by temperature changes.

Example 2

As a proof of principle, the responsive property of a macromolecularGd(III) complex towards pH, an other important diagnostic in vivo markerhas been investigated.

In this case, the slowly tumbling system chosen was the non covalentadduct formed by Gd-DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylene phosphonic acid) and poly-arginine. GdDOTP is anegatively charged macrocyclic complex able to bind, via electrostaticforces, to positively charged compounds, including cationic polyaminoacids like polyarginine. (Aime S, Angew Chem Int Ed 2003, 42,4527-4529).

The inner-coordination sphere of Gd-DOTP lacks of water molecules and,therefore, the relatively high relaxivity measured for this complex hasbeen accounted for in terms of a large second-sphere contributionarising from the presence of several water molecules tightly bound tothe phosphonate groups of the chelate and, therefore, dipolarly coupledto the Gd(III) ion.

In analogy to some other cationic polyamino acids like polylysine, thetumbling motion of polyarginine is pH dependent owing to the cross-overfrom a faster tumbling structure (at acidic pH) due to the repulsion ofthe positively charged guanidine groups to a more rigid and slowertumbling α-helix structure formed at basic pH when the guanidineresidues deprotonates.

The binding affinity between Gd-DOTP and polyarginine is so strong(K_(A)=3·10⁴ as determined by relaxometric mesurements) that it is veryeasy to prepare a solution in which the paramagnetic complex is fullybound to the polymer.

The R_(2p)/R_(1p) protocol for the pH measurement was tested on twosolutions containing different amounts of Gd-DOTP/polyarginine adduct(in 10 mM HEPES buffer). The concentration of the Gd(III) complex in thetwo solutions was 0.25 and 0.5 mM, respectively, and theGd-DOTP/polyarginine molar ratio was 10/1. R₁ and R₂ values for waterprotons were measured at 14.1 T (corresponding to a proton Larmorfrequency of 600 MHz) and 312 K.

The paramagnetic R_(1p) and R_(2p) contributions to the observedrelaxation rates were obtained by subtracting the correspondingdiamagnetic contributions (R_(id)) measured at each pH value in theabsence of the paramagnetic agent.

The result shown in FIG. 5 strongly support the right applicability ofthe proposed approach also to macromolecular systems not including anywater molecule(s) coordinated to the metal centre, provided that therelaxation rates of the said systems are dominated by the contributionof dipolarly coupled water protons present in the second coordinationsphere of the metal center.

Example 3

As a further proof of principle, the pH responsiveness of amacromolecular Gd(III)-based covalent adduct has been investigated.

The model system is represented by a Gd(III) chelate covalently linked,via a squaric moiety, to a poly-Ornithine backbone (Gd-II, FIG. 6).

As it has already discussed in the Example 2, it is known that basicpolyamino acids, like poly-Ornithine, undergo a pH-controlled reversibleconformational switch from a “random-coil” structure, predominant at pH<9 when the ornithine residues are protonated, to a “pseudo-α-helical”structure, predominant at pH >11 when the ornithine residues deprotonate(G C Hammes, P B Roberts, J Am Chem Soc, 91, 1812, 1969). Interestingly,it has been demonstrated that the rotational mobility of the twoconformations are different and a longer τ_(R) value has been observedfor the significantly more rigid and structurally ordered“pseudo-α-helical” conformation (see, for instance, S Aime, M Botta, S GCrich, G Giovenzana, G Palmisano, M Sisti, Chem Commun, 1577, 1999).

FIG. 7 reports the magnetic field dependence of R_(1p) and R_(2p)measured at three pH values, 7, 10, and 12, of a solution of Gd-IIcontaining 1 mM of Gd(III) ion. The R_(1p) enhancement observed uponincreasing pH is a clear evidence of the slowing-down of thereorientational motion of the macromolecolar complex caused by theconformational switch of the polymer. A detailed quantitative analysisof the R_(1p) profiles, performed by using theSolomon-Bloembergen-Morgan theory, implemented with the Lipari-Zsabomodel, indicated that both global (τ_(R) ^(g)) and local (τ_(R) ^(l))reorientational times for the macromolecular adduct are pH-dependent(τ_(R) ^(g) of 1.0, 1.7, and 3.6 ns were obtained at pH 7, 10, and 12,respectively).

Interestingly, the R_(2p) values are not dependent on magnetic field,because they are dominated by τ_(C) and the latter parameter, in thiscase, is dominated by τ_(R). For this reason, R_(2p) values aresignificantly affected by pH of the solution.

To confirm the concentration-independence of the R_(2p)/R_(1p) ratio,the paramagnetic contributions to the relaxation rates have beenmeasured at 14 T, 25° C., and at four pH values (7, 8.5, 10, and 12), asa function of the concentration of Gd(III) ion. The result, reported inFIGS. 8 and 9, shows unambigously that the R_(2p)/R_(1p) ratio isindependent on the concentration of the macromolecular agent butdependent on the parameter of interest.

Example 4

The responsive properties towards temperature of a liposome entrapping aGd(III) complex have been investigated as an example of a nano-sizedresponsive system according to the invention. The paramagnetic liposomehas been prepared by hydrating a thin lipidic film made of a mixture oflipids (POPC/Chol/DSPE-PEG, molar ratio 55:40:5 wherein POPC is1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine, Chol is Cholesterol,and DSPE-PEG is1,2-Distearoyl-sn-Glycero-3-Phosphoethanol-amine-N-(PolyethyleneGlycol)-2000)with a solution 200 mM of Gd-HPDO3A (a contrast agent marketed asProHance®). Upon repeated extrusion with a filter of 400 nm of diameter,the liposome suspension has been exhaustively dialysed against isotonicHepes buffer in order to remove the untrapped Gd(III) complex. The meanliposome diameter after dialysis was of 260 nm.

FIG. 10 reports the temperature dependence of R_(2p) and R_(1p) for thisnano-sized system, measured at 7 T. The two paramagnetic contributionsshow an opposite tendency upon increasing temperature: R_(2p) decreases,whereas R_(1p) increases.

The observed behaviour can be explained by considering that R_(1p)contribution is controlled by the water permeability of the liposomemembrane and, for this reason, a temperature increase lead to a Ripenhancement. On the contrary, magnetic susceptibility effects, whoseextent decrease as the temperature increases, dominate R_(2p) values.

The concentration-independence of the R_(2p)/R_(1p) ratio has beendemonstrated by measuring R_(2p) and R_(1p) values of solutionscontaining different amounts of liposomes (and consequently of Gd(III)complex, FIG. 11).

The different behaviour displayed by R_(1p) and R_(2p) versustemperature makes the R_(2p)/R_(1p) ratio strongly dependent on thisparameter (FIG. 12).

1. A method for the in vivo determination, by use of the MRI technique,of a physical or chemical parameter of diagnostic interest in a human oranimal body organ, fluid or tissue, said method comprising:administering to said human or animal a diagnostically effective amountof a slow tumbling paramagnetic agent that is responsive tomicroenvironmental changes of said parameter through changes in theR_(2p)/R_(1p) ratio; recording a MRI; and calculating the R_(2p)/R_(1p)ratio responsive for said parameter.
 2. The method of claim 1 whereinthe slow tumbling paramagnetic agent is a macromolecular paramagneticmetal complex endowed with at least one labile water moleculecoordinated to the metal center and having a τ_(R) value ≧1 ns and aτ_(M) value from 0.01 to 1 μs.
 3. The method of claim 2 wherein τ_(R) ofthe macromolecular paramagnetic complex is from 1 to 10 ns.
 4. Themethod of claim 1 wherein the slow tumbling paramagnetic agent is anano-sized system entrapping a paramagnetic metal complex.
 5. The methodof claim 4 wherein the nano-sized system has a mean diameter ≧5 nm, andthe concentration of the entrapped complex is ≧5 mM.
 6. The method ofclaim 1, wherein the measured R_(2p)/R_(1p) ratio acts as a reporter ofa change in the parameter of interest.
 7. The method of any one ofclaims 2 or 4, wherein the metal of the paramagnetic complex or agent isa Lanthanide or a Transition metal ion.
 8. The method of claim 7 whereinthe metal ion is selected from Fe(²⁺), Fe(³⁺), Cu(²⁺), Cr(3+), Eu(³⁺),Dy(³⁺), La(³⁺), Yb(³⁺) or Mn(²⁺) and Gd(³⁺).
 9. The method of claim 8wherein the metal ion is Gd(³⁺).
 10. The method of any one of claims 2or 3 wherein the macromolecular paramagnetic metal complex is the(Gd-1)₄/Avidin adduct, or the Gd-DOTP/polyarginine adduct, or the Gd-IImacromolecular adduct.
 11. The method of any one of claims 4 or 5wherein the paramagnetic nano-sized system is a liposome entrappingGd-HPDO3A.
 12. The method of any one of claims 1, 2 or 4, wherein thephysical or chemical parameter of diagnostic interest is selected fromtemperature, pH, partial pressure of oxygen or carbon dioxide, specificion or metabolite concentration, specific enzymatic activity.
 13. Adiagnostic composition comprising at least one slow tumblingparamagnetic agent endowed with a τ_(R) value from 1 to 10 ns and aτ_(M) value from 0.01 to 1 μs and for which the rotational mobilityτ_(R) and/or the mean residence lifetime τ_(M) of the mobile protonsdipolarly coupled to their metal center and/or the longitudinalelectronic relaxation time τ_(1S) are dependent on the physical orchemical parameter of diagnostic interest, for use in determining thesaid parameter in a human or animal body organ or tissue, by use of MRImaging.
 14. A diagnostic composition according to claim 13 wherein theslow tumbling paramagnetic agent is a nano-sized system with a meandiameter ≧5 nm entrapping a paramagentic metal complex at aconcentration ≧5 mM for which the size of the system and/or the magneticsusceptibility effects are dependent on a parameter of diagnosticinterest.
 15. A slow tumbling paramagnetic agent responsive to changesof a given physical or chemical parameter of diagnostic interest throughchanges in R_(2p)/R_(1p) ratio for use in the determination of the saidparameter in a human or animal body organ, fluid or tissue, by use ofthe MRI, in a manner which is independent on the absolute concentrationof said administered agent.
 16. A slow tumbling paramagnetic agentaccording to claim 15 comprising a macromolecular paramagnetic metalcomplex endowed with both dipolarly coupled protons and τ_(R) value ≧1ns, and wherein the rotational mobility τ_(R) and/or the mean residencelifetime τ_(M) of the mobile protons dipolarly coupled to their metalcenter and/or their longitudinal electronic relaxation time τ_(1S) aredependent on a physical or chemical parameter of diagnostic interest.17. A slow tumbling paramagnetic agent according to claim 15 comprisinga nano-sized system entrapping a paramagnetic metal complex for whichthe transverse relaxation rate is affected by magnetic susceptibilityeffects and for which the size and/or the extent of the magneticsusceptibility are dependent on a physical or chemical parameter or aphysiological or metabolic process of diagnostic interest.
 18. A slowtumbling paramagnetic agent according to claim 16, wherein the metal ionis gadolinium, having a τ_(R) value from 1 to 10 ns and a τ_(M) from0.01 to 1 μs.
 19. A compound selected from the following: the(Gd-1)₄/Avidin adduct wherein Gd-1 has the following structure

the macromolecular adduct Gd-II.
 20. The compound of claim 19 for use asa contrast agent.